The transformation of differentiated cells to induced pluripotent stem cells (iPSCs) has revolutionized stem cell biology by providing a more tractable source of pluripotent cells for regenerative therapy. The derivation of iPSCs from numerous normal and diseased cell sources has enabled the generation of stem cells for eventual use in cell therapy and regenerative medicine.
Seminal studies by Yamanaka and colleagues revealed that ectopic expression of certain transcriptional factors could induce pluripotency in somatic cells. These induced pluripotent stem cells self-renew and differentiate into a wide variety of cell types, making them an appealing option for disease- and regenerative medicine therapies. They have been used to successfully model human disease and have great potential for use in drug screening and cell therapy. Furthermore, iPSCs generated from diseased cells can serve as useful tools for studying disease mechanisms and potential therapies. However, much remains to be understood about the underlying mechanisms of reprogramming of somatic cells to iPSCs, and there is concern regarding potential clinical applications in the absence of mechanistic insights.
The original set of factors (RFs) for reprogramming to pluripotency include Oct3/4, Sox2, c-Myc, Klf4, Lin28, and Nanog. Oct3/4 and Sox2 are transcription factors that maintain pluripotency in embryonic stem (ES) cells while Klf4 and c-Myc are transcription factors thought to boost iPSC generation efficiency. The transcription factor c-Myc is believed to modify chromatin structure to allow Oct3/4 and Sox2 to more efficiently access genes necessary for reprogramming while Klf4 enhances the activation of certain genes by Oct3/4 and Sox2. Nanog, like Oct3/4 and Sox2, is a transcription factor that maintains pluripotency in ES cells while Lin28 is an mRNA-binding protein thought to influence the translation or stability of specific mRNAs during differentiation. It has also been shown that retroviral expression of Oct3/4 and Sox2, together with co-administration of valproic acid, a chromatin destabilizer and histone deacetylase inhibitor, is sufficient to reprogram fibroblasts into iPSCs.
Several classes of vectors have been shown to induce pluripotency when overexpressing the requisite gene combinations. The earliest vectors relied on DNA-integrating retroviruses and transposons for nuclear reprogramming. While effective, they inherently raise concerns about potential tumorigenicity either by insertional mutagenesis or re-expression of oncogenic reprogramming factors. While Cre-LoxP site gene delivery or PiggyBac transposon approaches have been used to excise foreign DNA from the host genome following gene delivery, neither strategy eliminates the risk of mutagenesis because they leave a small insert of residual foreign DNA.
As an alternative to genetic modification, mRNA, episomal DNA plasmids, and cell permeant proteins (CPP) have been shown to be effective for reprogramming factors.
These non-integrating vectors, however functional, often result in reduced reprogramming efficiencies either as a result of their specific mechanism of action or because of the cumbersome nature of their practice. Because, non-integrating and/or small-molecule based approaches for iPSC generation or transdifferentiation to a different somatic cell type are clinically relevant vectors, it becomes important to increase the robustness, efficiency, and ease of use of such methods. The present invention addresses these issues.